This invention relates to compositions and their methods of use in promoting the growth and/or regeneration of neurological tissue within the central nervous system (CNS).
CNS Damage
Approximately 1,100 new spinal cord injuries occur each year in Canada; over 10,000 per year occur in the United States. These numbers are five times higher if one also includes patients suffering brain damage involving inhibition to neural growth following traumatic brain injury. The number of patients with chronic spinal cord injuries in North America is in the order of 300,000. Again, this number is five times higher if one includes patients suffering from brain damage involving inhibition to neural growth following traumatic brain injury.
Spinal cord injuries often result in a permanent loss of voluntary movement below the site of damage. Mostly young and otherwise healthy persons become paraplegic or quadriplegic because of spinal cord injuries. There are an estimated 200,000 quadriplegics in the United States. Given the amount of care required, it is not difficult to envision how health care costs associated with caring for patients with central nervous system (CNS) damage is well over $10 billion a year for North America.
The CNS (the brain and the spinal cord) is comprised of neurons and glia, such as astrocytes, microglia, and oligodendrocytes. Neurons typically have two types of processes: dendrites, which receive synaptic contact from the axons of other neurons; and axons, through which each neuron communicates with other neurons and effectors. The axon of a CNS neuron is wrapped in a myelin sheath.
In higher vertebrates, axons within the CNS possess a limited capacity for repair after injury. Axotomized neurons of the adult mammalian CNS fail to exhibit substantial axonal regeneration, in contrast to neurons within the embryonic or neonatal CNS or within the adult peripheral nervous system (PNS) (Saunders et al., (1992) Proc. R. Soc. Lond. B. Biol. 250:171-180; Schwab and Bartoldi (1996) Physiol. Rev. 76:319-370; Steeves et al., (1994) Prog. Brain Res. 103:243-262). In fact, complete CNS axonal disruption is likely to preclude recovery. Although axotomized fibers proximal to the neuronal cell body initiate regenerative growth, this is subsequently aborted within a short distance (1-2 mm) and is often followed by retrograde degeneration. Although CNS axons will not regrow in the environment of the adult spinal cord, peripheral nerve grafts into the CNS provide a favorable environment through which CNS axons will anatomically regenerate (May et al., Cajal""s Degeneration and Regeneration of the Nervous System, History of Neuroscience Series #5 (NY and Oxford: Oxford Univ. Press, 1991) at 769). These findings indicate that adult CNS neurons retain intrinsic growth properties and, given favorable environmental conditions, are capable of successfully reactivating growth programs.
Current Treatments of Spinal Cord Injuries
A number of current therapies exist for the treatment of spinal cord injuries. Interventional therapies, including opiate antagonists, thyrotropin-releasing hormone, local cord cooling, dextran infusion, adrenergic blockade, corticosteroids, and hyperbaric oxygen have been utilized, but are of questionable clinical value.
Peripheral nerve transplants have been suggested as bridges across CNS lesions (David and Aguayo (1981) Science 214:931-933; Houle (1991) Exp. Neurol. 113:1-9; Richardson et al., (1984) J. Neurocytol. 13:165-182; Richardson et al., (1980) Nature 284:264-265; Xu et al., (1995) Exp. Neurol. 138:261-276; Ye and Houle (1997) Exp. Neurol. 143:70-81). Olfactory ensheathing cell transplants have been used recently to promote the regeneration of injured corticospinal projections in the rat (Li et al., (1997) Science 277:2000-2002). A recent study (Cheng et al., (1996) Science 273:510-513) employed a combinatorial approach that extended earlier work (Siegal et al., (1990) Exp. Neurol. 109:90-97): after transection of the adult rat spinal cord, peripheral grafts were used to connect white matter tracts to central gray matter in such a way as to direct regenerating fibers out of an inhibitory environment and into the more permissive gray matter. U.S. Pat. No. 5,650,148 and 5,762,926 describe a method for treating damage to the CNS by grafting donor cells into the CNS that have been modified to produce molecules such as neurotrophins.
The use of transplanted neural cells is also of limited clinical value: although axons will be able to grow into the transplanted tissue, they will not be able to grow out of the transplanted tissue back into the CNS due to inhibitory matter in the CNS.
This review of current methods of treating spinal cord injuries indicates that a need remains for a means of promoting regrowth, repair, and regeneration of neurons in the mammalian CNS in both the acute and chronic situations.
Myelin
It has been suggested that the failure of CNS axons to regenerate after injury is associated with the presence of myelin. The myelin sheath wrapping an axon is composed of compacted plasma membranes of Schwann cells and oligodendrocytes. Although its composition resembles that of any other plasma membrane in that it contains lipids, proteins, and water, the relative proportions and dispositions of these components are unique to myelin. Myelin in the CNS is produced by oligodendrocytes and is characterized by the expression of myelin basic protein (MBP). MBP is only associated with myelin and is one of the first proteins expressed at the onset of myelination of CNS axonal fibers. Galactocerebroside (GalC) is the major sphingolipid produced by oligodendrocytes. GalC comprises approximately 15 percent of the total lipid in human myelin and is highly conserved across species. Although GalC is expressed on the surface of oliogodendrocyte cell bodies, it is expressed in greater concentration on the surface of myelin membranes (Ranscht et al., (1982) Proc. Natl. Acad. Sci. USA 79:2709-2713).
There is growing evidence that the presence of CNS myelin can retard or inhibit the regenerative growth of some severed CNS axons (Schwab and Bartoldi (1996) Physiol. Rev. 76:319-370), including a number of examples from widespread vertebrate families (Schwegler et al., (1995) J. Neurosci. 15:2756-2767; Steeves et al., (1994) Prog. Brain Res. 103:243-262). Both the lower vertebrate CNS (e.g. lamprey) and the developing CNS of higher vertebrates (e.g. birds and mammals) exhibit substantial axonal regeneration after injury (Davis and McClellan (1994) J. Comp. Neurol. 344:65-82; Hasan et al., (1993) J. Neurosci. 13:492-507; Hasan et al., (1991) Restor. Neurol. Neurosci. 2:137-154; Iwashita et al., (1994) Nature 367:167-170; Saunders et al., (1992) Proc. R. Soc. Lond. B. Biol. 250:171-180; Treheme et al., (1992) Proc. Natl. Acad. Sci. USA 89:431-434; Varga et al., (1995) Eur. J. Neurosci. 7:2119-2129). The common phenotype for all these positive examples of regeneration is either a CNS that lacks compact myelin (lamprey) or incomplete myelin development (embryonic chick, neonatal opossum and rat) at the time of injury. The developmental appearance of myelin temporally correlates with the loss of regeneration by injured CNS axons. In addition, the robust growth of transplanted fetal neurons in the adult CNS (Bregman et al., (1993) Exp. Neurol. 123:3-16; Li and Raisman (1993) Brain Res. 629:115-127; Yakovleff et al., (1995) Exp. Brain Res. 106:69-78) may be partially attributed to either a lack of receptors for myelin inhibitors at that stage of their differentiation and/or an ability to override any inhibitory signals from myelin. Specific molecules associated with myelin have been identified as putative mediators of this inhibitory activity, including myelin-associated glycoprotein (MAG) (McKerracher et al., (1994) Neuron. 13:805-811; Mukhopadhyay et al., (1994) Neuron. 13:757-767) and NI35/250, an as yet unidentified myelin-derived protein (Bandtlow and Schwab (1991) Soc. Neurosci. Abstr. 17:1495; Caroni and Schwab (1988) J. Cell Biol. 106:1281-1288; Caroni and Schwab (1988) Neuron 1:85; Crutcher (1989) Exp. Neurol. 104:39-54; Savio and Schwab (1989) J. Neurosci. 9:1126-1133; Schwab and Caroni (1988) J. Neurosci. 8:2381-2393); IN-1 (Brosamle, et al, (1998) Abst. Soc Neurosci., 24:1559; NI-35/250 (Huber et al., (1998) Abst. Soc Neurosci., 24:1559; NI-220/250 (van der Haar et al., (1998) Abst. Soc Neurosci., 24:1559; arretin, Janani et al., (1998) Abst. Soc Neurosci., 24:1560; and NOGO (Chen et al., (1998) Abst. Soc Neurosci., 24:1776.
Experimental attempts to functionally block myelin-associated inhibition involving NI35/250, by using an anti-NI35/250 antibody, IN-1, have facilitated some anatomical regeneration of corticospinal axons (Bregman et al., (1995) Nature 378:498-501; Caroni and Schwab (1988) Neuron. 1:85-96; Schnell and Schwab (1990) Nature 343:269-272).
The immunological disruption of mature myelin within the avian spinal cord (Keirstead et al., (1995) J. Neurosci. 15:6963-6974), and the delay of onset of CNS myelination during normal avian or mammalian neurodevelopment (Keirstead et al., (1992) Proc. Natl. Acad. Sci. (USA) 89:11664-11668; Keirstead et al., (1997) Brain. Res. Bull. 44:727-734; Varga et al., (1995) Eur. J. Neurosci. 7:2119-2129) have also facilitated CNS axonal re-growth and/or sprouting.
The presence of certain components located or embedded in myelin that are inhibitory to the regeneration of axonal growth after injury makes it desirable to transiently remove myelin and its inhibitory components to promote the repair of injured adult spinal cord. Adult spinal cord can be demyelinated in vivo via drugs (e.g. ethidium bromide); however, these drugs have non-specific deleterious effects on other cell types in the central nervous system (e.g., astrocytes). In addition, myelin-deficient strains of mice and rats are readily available, but are of limited experimental value due to a shortened life span: most do not survive beyond a couple of weeks after birth.
Consequently, there is a need for improved methods of disrupting myelin in vivo in order to enhance regeneration of neurological tissue. The present invention provides methods that address this need.
Complement
The complement system is the primary humoral mediator of antigen-antibody reactions. It consists of at least 20 chemical and immunologically distinct serum proteins capable of interacting with one another, with antibody, and with cell membranes (see, for example, J. Klein, Immunology: The Science of Self-Nonself Discrimination (New York: John Wiley and Sons, 1982) at 310-346). The principal actors in this system are 11 proteins, designated C1 to C9, B, and D, which are present normally among the plasma proteins. These proteins are normally inactive, but they can be activated in two separate ways: the classical pathway or the alternate pathway.
The classical pathway is activated by an antigen-antibody reaction: when an antibody binds with an antigen, a specific reactive site on the constant portion of the antibody becomes activated, which in turn binds directly with the C1 molecule of the complement system. This sets into motion a cascade of sequential reactions, beginning with the activation of the C1 proenzyme. Only a few antigen-antibody combinations are required to activate many molecules in this first stage of the complement system. The C1 enzymes then activate successively increasing quantities of enzymes in the later stages of the complement system. Multiple end-products are formed, which cause important effects that help to prevent damage by an invading organism or toxin, including opsonization and phagocytosis, lysis, agglutination, neutralization of viruses, chemotaxis, activation of mast cells and basophils, and inflammatory effects.
The complement system can also be activated by an alternate pathway without the intermediation of an antigen-antibody reaction. Certain substances react with complement factors B and D, forming an activation product that activates factor C3, setting off the remainder of the complement cascade; thus, essentially all the same final products of the system are formed as in the classical pathway, causing the same effects. Since the alternate pathway does not involve an antigen-antibody reaction, it is one of the first lines of defense against invading microorganisms.
Since components of both the classical pathway and the alternative pathway of the complement system act locally to activate C3, this is the pivotal component of complement. C3 is a 195 kD protein, which comprises two disulfide bridged chains of 105 and 75 kD. The enzymatically active C4-C2 complex, activated in the classical pathway by the binding of C1q to an antigen-antibody complex, cleaves C3 into two fragments, C3a and C3b. The larger fragment, C3b, binds covalently to the surface of a target cell where it acts as a protease to catalyze the subsequent steps in the complement cascade. It is also recognized by specific receptor proteins on macrophages and neutrophils that enhance the ability of these cells to phagocytose the target cell. In particular, membrane-immobilized C3b triggers a further cascade of reactions that leads to the assembly of membrane attack complexes from the late components.
Complement fixation by cell-surface binding antibodies has been shown to compromise the ionic homeostasis of many different cells in vitro within minutes of activation (Mayer (1972) Proc. Natl. Acad. Sci. USA 69:2954-2958; Morgan (1989) Biochem. J. 264:1-14).
Use of Complement with Myelin-Specific Antibodies
After attachment of a specific complement-fixing antibody to a myelin surface antigen, serum complement forms a membrane attack complex through an enzymatic cascade resulting in a rapid influx of extracellular calcium (Dyer and Benjamins (1990) J. Cell Biol. 111:625-633) and subsequent cytoskeletal re-arrangement (Dyer and Matthieu (1994) J. Neurochem. 62:777-787). In vivo, this would make the disrupted myelin processes a target for phagocytosis by subsequent microglia, as well as by any invading macrophages.
The in vitro application of serum complement with myelin-specific antibodies has been shown to suppress myelin elaboration in purified oligodendrocyte cultures (Dorfinan et al., (1979) Brain Res. 177:105-114; Dubios-Dalcq et al., (1970) Pathol. Eur. 5:331-347; Dyer and Benjamins (1990) J. Cell Biol. 111:625-633; Fry et al., (1974) Science 183:540-542; Hruby et al., (1977) Science 195:173-175).
In vivo myelin disruption has been shown in the guinea pig optic nerve using anti-GaIC serum and complement (Sergott et al., (1984) J. Neurol. Sci. 64:297-303); myelin disruption was observed within 1 to 2 hours of treatment.
The Chick Model
In the avian model, the onset of myelination in the embryonic chick spinal cord at E13 coincides with the transition from a permissive to a restrictive period for the functional repair of transected spinal cord. The first appearance of chick oligodendrocytes on the tenth and eleventh embryonic day of development (E10-E11) precedes the initial formation of myelin by 2-3 embryonic days and is characterized by the expression of galactocerebroside (GalC), the major sphingolipid produced by oligodendrocytes.
In the mature avian spinal cord, after spinal cord transection, immunological disruption of local spinal cord myelin facilitated regeneration by brainstem-spinal neurons (Keirstead et al., (1995) J. Neurosci. 15:6963-6974; Keirstead et al., (1997) Brain Res. Bull., 44: 727-734). The immunological disruption of myelin was transient, produced by an intraspinal infusion of both serum complement and a myelin-specific, complement-fixing antibody (e.g. GalC antibodies). Such treatment resulted in the regeneration of up to 20% of mature brainstem-spinal axons.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention. Publications referred to throughout the specification are hereby incorporated by reference in their entireties in this application.
The purpose of the present invention is to provide a means of promoting regrowth, repair, and regeneration of neurons in the mammalian CNS. Accordingly, the invention provides compositions and methods of use for promoting regrowth, repair, and/or regeneration of neurons in the CNS of a mammalian subject, such as a human, in both chronic and acute disorders
One embodiment of the present invention provides a composition comprising therapeutically effective amounts of the following:
(a) one or more complement-fixing antibodies or fragments thereof, which specifically bind to an epitope of myelin; and
(b) one or more complement proteins or fragments thereof;
wherein the binding of said antibodies to myelin causes transient disruption and/or transient demyelination of myelin. The antibodies may be monoclonal and/or polyclonal. The complement proteins or fragments thereof may be derived from a species different from that species to which it is administered. In a preferred embodiment, the complement proteins or fragments thereof are human. The complement component may be a physically distinct component from the antibody component, or it may be covalently or noncovalently attached directly to the antibody component, such that binding of the antibody to the surface of the myelin triggers the endogenous immune system attack. One or more growth factors may be added (in an appropriate sequence) to facilitate regrowth and regeneration.
In a specific embodiment, the epitope of myelin is a myelin sheath epitope, such as galactocerebroside (GalC), O4, Myelin Oligodendrocyte Glycoprotein (MOG), or Myelin Associated Glycoprotein (MAG), NOGO, NI22, NI-35/250, or arretin, or fragments thereof. In a preferred embodiment, the epitope of myelin is GalC. Another preferred embodiment is MOG.
In a preferred embodiment, the complement proteins or fragments thereof include the C3 component or a fragment, variant, analog, or chemical derivative thereof. In a preferred embodiment, the component C3b is used.
In another embodiment of the present invention, the composition further comprises neurotrophins and growth factors, such as NT-3, CNTF, FGF-1, BDNF, PDGF, GDNF, CT-1, or BNP.
The present invention also relates to the use of these compositions to promote regrowth, repair, and/or regeneration of neurons in a subject by the transient disruption and/or transient demyelination of myelin.
In one embodiment of the present invention, the compositions are used in subjects requiring neuron repair and/or regeneration due to neuron dysfunction. This neuron dysfunction may be a result of acute or chronic injury to the CNS. It may also be a result of degenerative disease, such as Alzheimer""s or Parkinson""s disease.
In another embodiment of the present invention, the compositions are used in subjects to generate an environment within the CNS that is relatively permissive to growth of transplanted cells.
The present invention also relates to a method of promoting regrowth, repair, and regeneration of neurons in mammalian CNS, wherein the damage resulted from either a chronic or acute disorder. The method entails delivery of one or more complement-fixing antibodies or fragments thereof, which specifically bind to an epitope of myelin and delivery of one or more complement proteins or fragments thereof, delivered either together or separately to effect transient disruption and/or transient demyelination of myelin in the neuronal zone requiring regeneration.
Various other objects and advantages of the present invention will become apparent from the detailed description of the invention.